Mechanistic Studies of a Skatole-Forming Glycyl Radical Enzyme Suggest Reaction Initiation via Hydrogen Atom Transfer

Gut microbial decarboxylation of amino acid-derived arylacetates is a chemically challenging enzymatic transformation which generates small molecules that impact host physiology. The glycyl radical enzyme (GRE) indoleacetate decarboxylase from Olsenella uli (Ou IAD) performs the non-oxidative radical decarboxylation of indole-3-acetate (I3A) to yield skatole, a disease-associated metabolite produced in the guts of swine and ruminants. Despite the importance of IAD, our understanding of its mechanism is limited. Here, we characterize the mechanism of Ou IAD, evaluating previously proposed hypotheses of: (1) a Kolbe-type decarboxylation reaction involving an initial 1-e– oxidation of the carboxylate of I3A or (2) a hydrogen atom abstraction from the α-carbon of I3A to generate an initial carbon-centered radical. Site-directed mutagenesis, kinetic isotope effect experiments, analysis of reactions performed in D2O, and computational modeling are consistent with a mechanism involving initial hydrogen atom transfer. This finding expands the types of radical mechanisms employed by GRE decarboxylases and non-oxidative decarboxylases, more broadly. Elucidating the mechanism of IAD decarboxylation enhances our understanding of radical enzymes and may inform downstream efforts to modulate this disease-associated metabolism.


Comparative Genomics
At the beginning of our work, four

S20
To prepare chemically competent cells for plasmid transformations, the appropriate E. coli strain was cultured from a single colony in 100 mL LB broth, harvested at OD600 of 0.4-0.5, and resuspended in 10 mL of sterile Transformation-Storage Solution (10% w/v PEG 6000, 5% v/v DMSO, 20 mM MgCl2 in LB broth). 100 µL aliquots were flash frozen in liquid nitrogen and stored at -80 °C for later use. Plasmid transformations were performed by adding one or more plasmid (100 ng), 20 µL 5× KCM solution (0.5 M KCl, 0.15 M CaCl2, 0.25 M MgCl2), and 80 µL sterile water to a thawed competent cell aliquot of the appropriate strain. After 10 min incubation on ice, cells were heat shocked at 42 °C for 90 s, diluted to 1 mL with LB broth, and incubated at 37 °C for 1 h before plating with sterile beads on LB agar containing appropriate antibiotics.

Plasmid construction
Plasmid construction was carried out using standard molecular biology techniques. Descriptions of strains and plasmids are listed in Table S2AB. PCR amplifications were carried out with Q5 High-Fidelity or Phusion High-Fidelity polymerase, following manufacturer instructions. Primers (Sigma-Aldrich) are listed in  Site-directed mutagenesis of Ou IAD. pET28a-OuIAD(G853A) was constructed by amplifying the Ou IAD gene in two segments using primer sets pET28a_FP & OuIAD(G853A)_RP and OuIAD(G853A)_FP & pET28a_RP. The two segments were inserted into pET28a digested with NdeI and XhoI using the Gibson Assembly. 20 Other Ou IAD mutant constructs (C500S, H514A, H514E, E502Q, R226E, R226M, R226K, L616E, F401A, W392F, W392A) were constructed using a similar strategy.

A. E. coli strains
Dr. Benjamin J. Levin 5 Expression and purification of His6-tagged Ou IAD-AE, Cs HPAD-AE, and Cs HPAD enzymes. Ou IAD-AE, Cs HPAD-AE, and Cs HPAD were overexpressed and purified based on a reported protocol. 22 Chemically competent E. coli BL21(DE3) (CodonPlus-RIL) ∆iscR was transformed with the appropriate pET28a expression vector. An overnight culture of the appropriate strain was grown in LB media containing 50 µg mL -1 Km and 50 µg mL -1 chloramphenicol (Cm), starting from a colony or glycerol stock. 2 L of LB media supplemented with 20 mM MgCl2 • 6 H2O in a lightly screw-capped 2.8 L baffled Fernbach flask (Corning) was allowed to equilibrate overnight at 37 °C. Just prior to inoculation with 40 mL of starter culture (2%), the media was supplemented with 20 mM glucose and 50 µg mL -1 of Km and Cm. The culture was grown at 37 °C at 180 rpm to OD600 = 0.5-0.6, at which point 1 mM L-cysteine • HCl and 500 µM (NH4)2Fe(II)(SO4)2 • 6 H2O were added. Cultures were cooled on ice for 20 min and induced with 500 µM IPTG. The flask was immediately tightly sealed with a septa and screw cap before growing overnight at 16 °C at 180 rpm. Cell pellets were harvested aerobically by centrifugation at 6,730 × g for 10 min at 4 °C, flash frozen in N2 (l), and stored at -80 °C.

Glycyl radical quantification by electron paramagnetic resonance (EPR) spectroscopy
Much effort went into optimizing conditions for glycyl radical installation on Ou IAD by Ou IAD-AE. In brief various reductants (sodium dithionite ± methyl viologen, titanium citrate ± methyl viologen, acriflavine ± halogen lamp exposure, sodium ascorbate), length of halogen lamp illumination (0-2 h), buffer components (buffer identity, pH, NaCl concentrations), IAD:IAD-AE ratios, and FeS reconstitution of IAD-AE. 22 None of these optimizations improved Ou IAD activity relative to the conditions described below.
Wild-type Ou IAD and mutants were prepared for EPR spectroscopy as follows. All assay concentrations are final concentrations. Anoxic Ou IAD and Ou IAD-AE aliquots were brought into an anaerobic chamber containing N2 and < 0.  10.06 s; receiver gain, 52 dB. Simulated spectra were integrated twice to quantify the number of spins in each sample. All EPR assays were performed in triplicate.
The sulfide content of a 6 µM solution of Ou IAD-AE was determined using a previously published procedure, 29 with the only differences being that assays were performed in microcentrifuge tubes, assay volumes were doubled, pipetting was performed instead of stirring, and the mixture was incubated for 20 min after the addition of NaOH.
To obtain UV-vis spectra, Ou IAD-AE was diluted to 25 µM with anoxic buffer (50 mM HEPES, 50 mM NaCl, pH 8.0) inside of an anaerobic chamber at 4 °C containing 97% N2 and 3% H2 (CoyLabs). The absorbance of the solution was measured from 200 nm to 1200 nm in a septa-sealed Ultra-Micro Cell quartz cuvette (Hellma) using a Cary 8454 UV-Vis Diode Array System (Agilent). To obtain a spectrum for the reduced protein, 100 µM sodium dithionite was added with a gas-tight syringe before the absorbance was measured at 1, 4, 10, 25, and 30 min.  Table S3 for specific detection parameters. For quantification, standards of 5′-dA, SAH, and MTA were prepared ranging from 50-600 µM in H2O, in triplicate.  Table S3 for specific detection parameters. For quantification, standards were prepared ranging from 50-600 µM in H2O, in triplicate. HPLC assay for detecting I3A, skatole, and I3A analogs HPLC samples were prepared in the same way as the UPLC-MS samples for detecting SAM cleavage products except that they were subsequently quenched by the addition of 17 µL of 5% HPLC-grade trifluoroacetic acid (TFA) in LC-MS grade acetonitrile. The supernatant was analyzed by HPLC on an Inspire C18 column (5 µm particle size, 50 × 4.6 mm) (Dikma Technologies). 20 µL of each sample was injected onto the column. The flow rate was 1 mL min -1 using 0.1% formic acid in water as mobile phase A and 0.1% formic acid in acetonitrile as mobile phase B. The column was maintained at 25 °C. The following gradient was applied: 0-3 min: 5% B isocratic, 3-10 min: 5-90% B, 10-12 min: 90% B isocratic, 12-3 min: 90-5% B, 13-16 min: 55% B isocratic. All compounds were detected by measuring the absorbance at 280 nm. See Table S4 for specific detection parameters. For quantification, standards were prepared ranging from 50-600 µM in H2O, in singlicate.
Kinetic isotope effects (KIEs) from competition assays were determined using the following equation 30 : The KIE of each biological triplicate was determined when at least half of the substrate was consumed and then averaged. Assays were repeated on two independent days with similar results (Figure S8).

UPLC-MS/MS assay for IAD incubations in D2O
50 µM Ou IAD was activated as previously described. Assays were performed in PCR strips (VWR International) containing pre-aliquoted solutions of 1 mM D0-I3A, D2-I3A, or skatole in various ratios of buffer (50 mM HEPES [8], 50 mM NaCl) in H2O:D2O. Activated IAD was added (2.5 µM final) and the reactions were incubated for 4.5 hr. The sealed PCR strips were taken out of the anaerobic chamber, quenched with 5 equivalents of LC-MS grade acetonitrile containing 50 µM D7-I3A and 50 µM indole internal standards and analyzed by UPLC-MS/MS as previously described.
Analysis of hydroxyphenylacetate and p-cresol was carried out on a Thermo Scientific Dionex UltiMate 3000 UHPLC coupled to a Thermo Q Exactive Plus mass spectrometer system (Thermo Fisher Scientific Inc, Waltham, MA) equipped with an APCI probe for the Ion Max API source. Data were acquired with Chromeleon Xpress software for UHPLC and Thermo Xcalibur software version 3.0.63 for mass spectrometry and processed with Thermo Xcalibur Qual Browser software version 4.0.27.19.
The MS conditions were as follows: negative ionization mode; scan range, 85-165 m/z; resolution, 140,000; AGC target, 1e6; maximum IT, 480 ms; spray voltage, 5000 V; capillary temperature, 325 °C; sheath gas, 28; Aux gas, 5; maximum spray current, 5; probe heater temperature, 432 °C; S-Lens RF level, 55.00. A mass window of ± 5 ppm was used to extract the ion of [M-H] -for the compounds. Targets were considered detected when the mass accuracy was less than 5 ppm and there was a match of isotopic pattern between the observed and the theoretical ones and a match of retention time between those in real samples and standards.

Computational details
Electronic structure calculations were performed to investigate the energetics of substrates, intermediates, and products along the hypothesized intrinsic reaction pathways (i.e., without explicitly modeling the enzyme environment) using ORCA      Table S7.

Synthesis of α-methyl-indole-3-acetic acid and D3-skatole
All chemicals and solvents were purchased from Sigma-Aldrich unless otherwise noted. Anhydrous reactions were performed using oven dried or flame-dried glassware, which were then cooled under vacuum and purged with nitrogen (N2) gas. Unless otherwise noted, all proton nuclear magnetic resonance ( 1 H NMR) spectra and carbon nuclear magnetic resonance ( 13 C NMR) spectra were recorded on a Bruker AVANCE NEO 400 (400 MHz, 100 MHz) NMR spectrometer.
Mass spectrometry data were obtained using an Agilent 6530 quadruple time-of-flight mass spectrometer with an ESI source. The mass spectra data were recorded on positive ionization mode with a mass range of 100 to 3000 m/z; spectra rate, 10 spectra s -1 ; capillary voltage, 4500 V; nebulizer pressure, 22 psi; drying gas (N2) flow, 8 L min -1 ; temperature, 200 ºC.